Investigators in Project 3 (M.K. Brenner, Leader) will seek to improve immunotherapy for neuroblastoma by genetically modifying Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (GTLs) to express receptors specific for GD2 and FRAME, two antigens expressed at high levels on most neuroblastoma cells, and for CCL2 (MCP-1), a neuroblastoma-secreted chemokine. This strategy is supported by clinical and laboratory studies in Years 1-4 of this grant, demonstrating consistently superior in vivo survival and functionality for EBV CTLs expressing GD2 via a chimeric antigen receptor (CAR) compared to GD2-CAR+ primary T cells not specific for EBV. To enhance the trafficking of GD2-CAR+ EBV CTLs to neuroblastoma deposits, Dr Brenner's group proposes to express CCR2, the receptor for CCL2, on their modified CTLs and confirm its activity and safety in a xenograft tumor model (Aim 1). Then, in a Phase 1 clinical trial, they will administer two aliquots of CAR+ EBV CTLs - one with and one without CCR2 expression - to lymphodepleted patients with relapsed neuroblastoma, immediately after autologous stem cell transplantation, to determine the safety, in vivo expansion and persistence of CCR2- vs. CCR2+ CTLs, the infiltration of each type of CTL into tumor sites and the effects on residual tumor (Aim 2). Finally, they will prepare PRAME-specific alpha-beta TCRs from the T cells of immune patients, express them in EBV CTLs, test the safety and efficacy of the modified cells in animal models of PRAME/EBV+ tumors, and determine whether infusion of a combination of GD2-CAR+ and PRAME-specific alpha-beta TCR+ CTLs produces additive or synergistic effects (Aim 3). This plan of research, if successful, will overcome two major obstacles to adoptive immunotherapy for neuroblastoma (limited expansion and trafficking of CTLs) and will stimulate numerous interactions with others in the program who are circumventing the immune evasion tactics of EBV-positive tumors (Projects 1 and 4) or modulating the tumor microenvironment to allow optimal T-cell function (Projects 2 and 4) Lay summary- The effectiveness of cancer immunotherapy is limited by many factors, including the inability of the immune cells to migrate to deposits of tumor cells and expand their numbers at the tumor site. This group will test several promising strategies that may by- pass these obstacles, leading to greater destruction of neuroblastoma cells by activated immune lymphocytes.